Mammalian spermatogenesis is supported by a robust stem cell system that exquisitely balances self renewal with differentiation. Spermatogonial stem cells (SSCs) and their progenitors lie on the basal side of the Sertoli cell tight junctions and therefore outside the blood-testis barrier making them readily accessible targets for contraceptive intervention. Cells that manifest stem cell properties reside within a population of cells referred to as undifferentiated spermatogonia (Aundiff)- Different cells within the Aundiff population contribute to the stem cell pool during steady state spermatogenesis than during regeneration. Our proposed studies address the regulation of local signaling within the stem cell niche as well as germ cell intrinsic factors that regulate stem cell self-renewal, proliferation and differentiation. We propose that heparin sulfate proteoglycans (HSPGs) within the extracellular matrix (ECM) and on the surface of SSCs regulate the bioavailability of glial cell line-derived neurotrophic factor (GDNF), a critical signaling molecule expressed by Sertoli cells that is required for stem cell self-renewal. Using a combination of live cell imaging of fluorescently labeled SSCs in seminiferous tubule explants, germ cell transplantation and gerietic analysis in vivo, we will test the hypothesis that GDNF regulates SSC self-renewal by acting as a chemoattractant to sequester SSCs from inductive differentiation signals. In preliminary studies we have established a critical role for LIN28A, a highly-conserved RNA binding protein and regulator of the let-7 class of miRNAs that is expressed in Aundif1 in regulating the size of the Aundiff compartment. We propose experiments to distinguish between a role for LIN28A in proliferation and differentiation of Aund/Yf spermatogonia. Lastly, to discover additional regulators of SSC self-renewal, we will use high-throughput sequencing of RNA (RNAseq) isolated from polysomes of SSCs, Sertoli cells and peritubular myoid cells obtained from testis that are blocked in the transition from Aal to A1 spermatogonia under conditions of steady-state and regenerative SSC self-renewal.